We have identified a new mode of EGF receptor (EGFR) ligand signaling via exosomes. We show that colorectal cancer (CRC) cells release exosomes that contain multiple, full-length, signaling-competent EGFR ligands. Exosomes from DLD-1 colon cancer cells with a mutant K-RAS allele exhibit both higher amphiregulin (AREG) levels and greater invasive potential than exosomes from isogenically matched, non-transformed cells in which mutant K-RAS was eliminated by homologous recombination. In addition to AREG, mutant KRAS, EGFR and GLUT1 are enriched in exosomes from cells with mutant K-RAS compared to WT K-RAS. Upon exposure of DKs-8 cells to DKO-1 exosomes, mutant KRAS is transferred to and transforms these non- transformed cells. We propose three aims to elucidate the role of AREG-containing exosomes in CRC and to explore the hypothesis that some of the cell non-autonomous effects of mutant KRAS may be mediated by exosomes. Aim 1. Compare the tumor-promoting biological activity of exosomal AREG to secreted, soluble AREG and determine the mechanism by which AREG is delivered to exosomes. We predict that exosomal AREG will be more potent than soluble AREG. We also predict AREG is delivered from the PM to MVB and that mono-ubiquitylation of AREG is a necessary post-translational modification in this process. Aim 2. Elucidate the tumor-promoting biological activity of exosomal AREG, mutant KRAS and GLUT1 on recipient cells in vitro and in vivo. We will assess the contribution of exosomal AREG and mutant KRAS from mutant KRAS-expressing donor cells to soft agar growth, invasion and prolonged AKT signaling for recipient non-transformed intestinal epithelial cell lines. We will test the ability of exosomal GLUT1, and possibly other intermediary metabolites, to transfer the Warburg effect to non-transformed intestinal cell lines. Aim 3. Determine whether exosomes containing EGFR act as a decoy for cetuximab in vivo and determine the level of EGFR and AREG in serum exosomes from CRC patients before and after treatment with cetuximab and other therapies.